The present invention is directed to a DNA construct which can be used for either direct or indirect gene therapy. The DNA constuct contains muscle specific regulatory elements and a DNA sequence which encodes an antigen for immunization or a protein for gene therapy or the DNA sequence is an antisense sequence for gene therapy.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the following text and respectively grouped in the appended List of References.
Initial efforts toward postnatal gene therapy have relied on indirect means of introducing new genetic information into tissues: target cells are removed from the body, infected with viral vectors carrying the new genetic information and then replanted into the body. (Ledley, 1987; Eglitis and Anderson, 1988; Friedmann, 1989). However, indirect gene transfer is not useful for many applications of gene therapy. In these instances, direct introduction of genes into tissues in vivo is desired.
Several gene transfer systems have been developed to directly or indirectly introduce genes into tissues in vivo. These systems include viral and nonviral transfer methods. A number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990), vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990; Srivastava, 1993), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freese et al., 1990), and retroviruses of avian (Brandyopadhyay and Temin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al., 1988), and human origin (Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990; Buchschacher and Panganiban, 1992). Most human gene therapy protocols have been based on disabled murine retroviruses.
Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); mechanical techniques, for example microinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster et al., 1981; Constantini and Lacy, 1981); membrane fusion-mediated transfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989; Kaneda et at, 1989; Stewart et al., 1992; Nabel et al., 1990; Lim et al., 1992); and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al., 1989b; Wolff et al., 1991; Wagner et al., 1990; Wagner et al., 1991; Cotten et al., 1990; Curiel et al., 1991a; Curiel et al., 1991b). Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viral vectors to the tumor cells and not into the surrounding nondividing cells. Alternatively, the retroviral vector producer cell line can be injected into tumors (Culver et al., 1992). Injection of producer cells would then provide a continuous source of vector particles. This technique has been approved for use in humans with inoperable brain tumors.
In an approach which combines biological and physical gene transfer methods, plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector. The trimolecular complex is then used to infect cells. The adenovirus vector permits efficient binding, internalization, and degradation of the endosome before the coupled DNA is damaged.
Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is nonspecific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration (Nabel, 1992).
Direct gene transfer into mammalian somatic tissues in vivo is a developing technology with potential applications in human gene therapy. The principal advantages of such an approach are the simplicity and safety of the techniques. Three types of direct gene transfer methodology have been developed: particle bombardment, liposome-mediated deliver and naked DNA transfer. In particle bombardment methods, first applied to the transformation of plant tissue (Klein et al., 1987), the DNA-coated particles are accelerated to high velocity so that they are able to penetrate target organs, tissues or single cells efficiently. Gene transfer to various mammalian somatic tissue has been effectively achieve in vito, ex vivo and in vitro with particle bombardment (Yang et al., 1990). Liposome-mediated gene transfer is also an effective method for in vivo gene transfer. For example, DNA-liposome complexes have been used for direct gene transfer to human melanoma cells (Nabel et al., 1993).
A challenge to the development of vaccines against viruses such as influenza A or human immunodeficiency virus (HIV), against which neutralizing antibodies are generated, is the diversity of the viral envelope proteins among different isolates or strains. Because CTLs in both mice and humans are capable of recognizing epitopes derived from conserved internal viral proteins (Townsend et al., 1989) and are thought to be important in the immune response against viruses (Taylor et al. 1986), efforts have been directed toward the development of CTL vaccines capable of providing heterologous protection against different viral strains. CD8.sup.+ CTLs kill virally infected cells when their T cell receptors recognize viral peptides associated with major histocompatibility complex (MHC) class I molecules (Germain, 1981). These peptides are derived from endogenously synthesized viral proteins, regardless of the protein's location or function in the virus. Thus, by recognition of epitopes from conserved viral proteins, CTLs may provide cross-strain protection. Peptides capable of associating with MHC class I molecules for CTL recognition originate from proteins that are present in or pass through the cytoplasm or endoplasmic reticulum (Yewdell et al. 1989). Therefore, in general, exogenous proteins, which enter the endosomal processing pathway (as in the case of antigens presented by MHC class II molecules), are not effective at generating CD8.sup.+ CTL responses.
Most efforts to generate CTL responses have either used replicating vectors to produce the protein antigen in the cell (Hahn et al. 1992) or have focused on the introduction of peptides into the cytosol (Collins et al. 1992). Both of these approaches have limitations that may reduce their usefulness as vaccines. Retroviral vectors have restrictions on the site and structure of polypeptides that can be expressed as fusion proteins and still maintain the ability of the recombinant virus to replicate (Miller et al. 1992), and the effectiveness of vectors such as vaccinia for subsequent immunizations may be compromised by immune responses against the vectors themselves (Cooney et al. 1991). Also, viral vectors and modified pathogens have inherent risks that may hinder their use in humans (Mascola et al. 1989). Furthermore, the selection of peptide epitopes to be presented is dependent on the structure of an individual's MHC antigens, and peptide vaccines may therefore have limited effectiveness due to the diversity of MHC haplotypes in outbred populations (Townsend et al. 1989; Taylor et al. 1986; Germain, 1981). Hence, immunization with nonreplicating plasmid DNA encoding viral proteins may be advantageous because no infectious agent is involved, no assembly of virus particles is required, and determinant selection is permitted. Because the sequence of nucleoprotein (NP) is conserved among various strains of influenza (Gammelin et al. 1989; Gorman et al. 1991), protection was achieved here against subsequent challenge by a virulent strain of influenza A that was heterologous to the strain from which the gene for NP was cloned. Vectors used vaccines have also been described by Kieny et al. (1992), Hock et al. (1993) and Yankaukas et al. (1993).
Intramuscular (i.m.) injection of DNA expression vectors in mice has been demonstrated to result in the uptake of DNA by the muscle cells and expression of the protein encoded by the DNA (Ascadi et al. 1991; Fazio et al., 1994). Plasmids were shown to be maintained episomally and did not replicate. Subsequently, persistent expression was observed after i.m. injection in skeletal muscle of rats, fish and primates, and in cardiac muscle of rats (Wolff et al. 1992).
Muscle creatine kinase (MCK) is expressed at high levels in both skeletal and cardiac muscle of adult animals (Eppenberger et al. 1964; Jockers-Wretou et al. 1975; Richterich et al. 1967; Tanzer et al. 1959). Activation of MCK transcription during skeletal myoblast differentiation has been shown (Chamberlain et al. 1985; Jaynes et al. 1986; Perriard 1979; Perriard et al. 1978; Rosenberg et al. 1982), and multiple cis-acting regulatory sequences have been identified in the 5' flanking sequence and first intron of the MCK gene (Jaynes et al. 1988; Sternberg et al. 1988). The best-characterized element is a 207-base-pair (bp) muscle-specific enhancer located about 1,100 nucleotides (nt) 5' of the MCK transcription start site. Another enhancer element is located within a 900-nt region in the first intron. The proximal 776-nt 5' MCK sequence also displays muscle cell type specificity in cultured cells, but the absolute level of expression from this element is quite low compared with expression when either enhancer is present (Jaynes et al. 1988). A myocyte-specific binding activity, MEF1, that interacts with both enhancers but not the proximal element, has been identified (Buskin et al. 1989). Furthermore, the intact MEF1 site is required for the 5' enhancer to function in MCK expression during skeletal myoblast differentiation in culture.